Fungal infection in the U.S. has increased significantly over the past three decades, especially among non-albicans species. While Candida albicans remains the primary species involved in Candida infections (40-70%), Candida glabrata is now recognized as the second most common cause (10-30%) and a true emerging pathogen (Ostrosky-Zeichner, L. et al. Antimicrob. Agents Chemother. 47:3149-3154, 2003). Like Candida albicans, Candida glabrata infections can occur in the oral and vaginal mucosal membranes, as well as the urinary tract, and often lead to life-threatening systemic and deep-seated infection within the immuno-compromised host. With its mortality rates up to 51%, the highest amongst the Candida species, Candida glabrata presents a serious threat to immuno-compromised patients (Komshian, S. V. et al. Rev. Infect. Dis. 11:379-390, 1989).
A common fungal infection in healthy women is vulvovaginal candidiasis (WC), which occurs in approximately 75% of women at least once in their lifetime, with 5 to 8% of those individuals developing a recurrent infection (Fidel, P. L. et al. Clin. Microbiol. Rev. 9:335-348, 1996). The epidemiological data on Candida species distribution involved in WC is limited; however, some studies report that Candida albicans accounts for 70-90% of all cases with a recent emergence in non-albicans species being observed. A recent study looking at a large collection of samples from women suspected of having WC found that non-albican species, particularly Candida glabrata, become more prevalent with an increase in age occurring around the time of menopause (Vermitsky, J.-P. et al. J. Clin. Microbiol., 2008). Candida glabrata is the primary non-albicans species emerging in WC, accounting for up to 14% of infections in immuno-competent women and the primary species isolated from diabetic patients (61.3%) and the elderly (51.2%). Several proposed factors may contribute to the emergence of non-albicans species, including the use of azole anti-fungals (e.g., both triazoles and over-the-counter imidazoles), changes in the patient's immune or physiological status, and an increase in the use of chemotherapeutic treatments.
Identifying the species and the antimicrobial susceptibility of an isolate involved in infection is imperative for determining the proper course of treatment. Surveillance programs performed over the past few decades have demonstrated that although azole resistance is rare in Candida albicans isolates (<1%), it is becoming very common among Candida glabrata (up to 15%) and other non-albicans species (Richter, S. S. et al., J. Clin. Micobiol. 45(5):2155-2162, 2005). Current practices for determining the susceptibility of a particular isolate involve the microbroth dilution assay (MBD) outlined by the Clinical Laboratory Standards Institute's (CLSI) M27-2A document (National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution anti-fungal susceptibility testing of yeasts; approved standard, 2nd ed., M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.) as well as several commercially available tests such as Sensititre® YeastOne (Trek Diagnostic Systems, Inc., Westlake, Ohio) and Etest® (AB BIODISK, Solna, Sweden). Although the MBD assay is quite effective, results can take upwards of a week to obtain. Additionally, for Candida glabrata, it was reported that upon comparison with the MBD, these commercially available tests could provide inaccurate or non-concordant susceptibility results, especially when testing with azole anti-fungals (Alexander, B. D. et al., J. Clin. Microbiol. 45(3):698-706, 2007). The need to develop a more rapid, yet still reliable, method for determining the susceptibility of yeast isolates is warranted.
A number of publications have shown that the predominant mechanism of azole resistance in Candida glabrata is the increased constitutive expression of their drug efflux pumps or multidrug resistance genes (MDR) (Miyazaki, H. et al., Antimicrob. Agents Chemother. 42:1695-1701, 1998; Sanglard, D. et al., Antimicrob. Agents Chemother. 43:2753-2765, 1999; Vermitsky, J.-P. et al., Antimicrob. Agents Chemother. 48(10):3773-3781, 2004). Recent publications have established qRT-PCR markers to test for azole resistance in Candida albicans (Kofla, G. et al., J. Microbiol. Methods. 68:178-183, 2007; Park, S. et al., Microbial. Drug Resist. 11(3):232-238, 2005). Due to the fact that resistance is far more prevalent in Candida glabrata than Candida albicans, this organism warrants greater attention when determining a treatment strategy. Moreover, Candida glabrata has the ability to develop frank resistance, which too often results in ineffective azole treatment on resistant (R) or susceptible dose-dependent (S-DD) Candida glabrata isolates and further necessitates azole susceptibility testing. The use of PCR-based identification of molecular markers for susceptibility testing can lead to a more rapid turn-around-time, allowing the physician to choose the proper course of treatment in a timely manner to improve patient outcome. The results of the qRT-PCR assay demonstrate differential expression of MDR genes within S-DD and resistant isolates suggesting different mechanisms between the two susceptibility states.
Despite these known gene expression patterns, though, an effective anti-fungal susceptibility assay with desirable sensitivity and specificity has not yet been described. Investigating this problem, the present inventors found that mere knowledge of the differential expression pattern was insufficient to provide such an assay. Assays that simply categorize isolates as resistant or susceptible based on conventional techniques and calculations often produce false positives and false negatives, each of which presents serious concerns for health care providers and public health officials. The basis for this failure in translating the knowledge into a useful and reliable test for years is unknown.
Accordingly, there is a continuing need for a reliable susceptibility assay for Candida glabrata that offers results in a relatively shorter time frame and high specificity and sensitivity. An improved assay would assist greatly in the detection of azole resistance in Candida glabrata infections from clinical samples, enabling health care providers to initiate proper treatment earlier in the course of the Candida infection.